The long-term objective of this research is to develop a clinically translatable strategy to repair soft tissue defects resulting from cancer, trauma, congenital abnormalities, and infections. Repair of soft tissue defects with tissue engineered fat grafts are the ideal solution to obviate many current limitations in reconstructive surgery. However, the fundamental question of how engineered fat grafts survive remains largely unanswered. The immediate objective of this proposal is to address three fundamental issues that hinder the progress of adipose tissue engineering. We hypothesize that successful grafting of engineered fat is linked to the cellular composition of the graft and the microenvironment of the graft's recipient site is tested. Three specific aims investigate this hypothesis utilizing an integrated, multidisciplinary approach. Aim 1 focuses on defining the cellular composition of syringe-aspirated human fat as a function of the techniques used to process harvested fat for implantation. Flow cytometry is employed to identify and quantify cell populations. In Aim 2, we determine the relationship between engineered fat graft volume and local blood supply. This critical design parameter is elucidated using quantitative histomorphometry and a xenograft model that incorporates a fat construct possessing its own pedicled blood supply. Aim 3 concentrates on determining the requisite role of the recruitment of resident fat precursor and stem cells. The development of an engineered fat graft will broadly impact health care related to soft tissue defects caused by cancer, trauma, congenital abnormalities, and infections. Understanding the cell populations involved in fat grafting and blood supply dependence of fat grafts are critical mechanisms that affect the design of an engineered fat graft. Moreover, the involvement of resident adipogenic cells at the site of fat grafting may modulate the survival of fat grafting. These fundamental issues, once understood, offer the potential to mimic or modulate these activities for novel bioinspired technologies. [unreadable] [unreadable] [unreadable]